Apoptotic entities for use in treatment of endothelium dysfunction disorders

ABSTRACT

Treatment and/or prophylaxis of endothelial dysfunction-related disorders in mammalian patients is effected by administering to the patient effective amounts of apoptotic bodies and/or apoptotic cells.

FIELD OF THE INVENTION

[0001] This invention relates to biochemical and biological compositionsand to the uses thereof in the treatment and/or prophylaxis, inmammalian patients, of various medical disorders associated withendothelial dysfunction (malfunctioning of the lining of blood vessels).More particularly, it relates to treatment and prophylaxis of medicaldisorders associated with endothelial dysfunction by administration ofcompositions containing mammalian cellular materials and fragmentsthereof, and to the compositions containing the mammalian cellularmaterials and fragments themselves, and to processes for preparing suchcompositions.

BACKGROUND OF THE INVENTION

[0002] Two mechanisms of cell death in the body are recognized, necrosisand apoptosis. Apoptosis is the process of programmed cell death,described by Kerr et al in 1992 [Kerr J F R, Wyllie A H, Currie A R(1992). “Apoptosis: a basic biological phenomenon with wide-rangingimplications in tissue kinetics. “British Journal of Cancer 26: 239-257”], by which steady-state levels of the various organ systems and tissuesin the body are maintained as continuous cell division anddifferentiation takes place. Cells undergoing apoptosis often exhibitdistinctive morphological changes such as a pronounced decrease in cellvolume, modification of the cytoskeletons resulting in pronouncedmembrane blebbing, a condensation of the chromatin, and degradation ofthe DNA into oligonucleosomal fragments. Following these morphologicalchanges, an apoptotic cell may break up into a number of small fragmentsknown as apoptotic bodies, comprising membrane-bound bodies containingintact organelles, chromatin etc. Apoptotic bodies are normally rapidlyremoved from the body by phagocytosis by macrophages, dendritic cellsand other antigen-presenting cells, before they can become lysed andrelease their potentially pro-inflammatory intracellular contents.

[0003] In simple outline, apoptosis is thought to proceed as follows.Three phases can be identified in the apoptotic mechanism of programmedcell death:

[0004] Induction phase;

[0005] Effector phase; and

[0006] Degradation phase.

[0007] The induction phase is dependent in part on specific interactionsof death-inducing signals at the cell surface membrane. One commonsignal is initiated by the binding of specific ligands to receptors ofthe TNF receptor family present on the cell membrane. One important suchreceptor is Fas (APO-1, CD95), which interacts with Fas-ligand toinitiate apoptosis.

[0008] The effector phase, activated by the binding of receptors andligands of the induction phase, leads to the activation of caspases,cystinyl-aspartate-requiring proteinases (proteolytic enzymes),including caspases 1 and 8. This activation may be associated with achange in the permeability of mitochondria, allowing the release ofcytochrome-c which is involved in caspase activation. Activated caspasesinitiate a chain of lethal proteolytic events culminating in the changesin chromatin and cytoskeletal components seen in apoptosis.

[0009] Many cells undergoing apoptosis can be identified by acharacteristic ‘laddering’ of DNA seen on agarose gel electrophoresis,resulting from cleavage of DNA into a series of fragments. These changesoccur a few hours before death of the cell as defined by the ability ofa cell to exclude vital dyes. The appearance of DNA laddering on agarosegel electrophoresis following extraction of DNA from cells is onerecognised method of identification of apoptosis in cells [Loo, D. T.and Rillema, J. R. (1998) “Measurement of Cell Death,” Methods in CellBiology 57: 251-264], although it is not always sensitive enough todetect apoptosis. In situ labelling of nuclear DNA fragmentation, forexample, using commercially available terminal dUTP nick end labelling(TUNEL) assays, are an alternative and more reproducible measure for thedetermination of fragmented DNA in apoptotic cells and cells undergoingapoptosis [Gavrieli Y, Sherman Y, Ben-Sasson S A (1992) “Identificationof programmed cell death in situ via specific labelling of nuclear DNAfragmentation,” Journal of Cell Biology 119: 493-501].

[0010] During apoptosis, phosphatidylserine becomes exposed externallyon the cell membrane [Fadok V A, Voelker D R, Campbell P A, Cohen J J,Bratton D L, Henson P M (1992), “Exposure of phosphatidylserine on thesurface of apoptotic lymphocytes triggers specific recognition andremoval by macrophages”. Journal of Immunology 148: 2207-2216] and thisexposed phosphatidylserine binds to specific receptors to mediate theuptake and clearance of apoptotic cells in mammals [Fadok V A, Bratton DL, Rose D M, Pearson A, Ezekewitz R A B, Henson P M (2000), “A receptorfor phosphatidylserine-specific clearance of apoptotic cells”, Nature405: 85-90]. The surface expression of phosphatidylserine on cells isanother recognised method of identification of apoptotic cells.

[0011] Changes in mitochondrial integrity are intimately associated withapoptosis, resulting in alterations in mitochondrial membranepermeability and the release of cytochrome-c from the mitochondria intothe cell cytoplasm [Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I,Brenner, C., Larochette, N., Prevost, M. C., Alzari, P. M. and Kroemer,G. (1999) “Mitochondrial Release of Caspase-2 and -9 during theApoptotic Process”, Journal of Experimental Medicine, 189: 381-394].Measurement of changes in mitochondrial membrane potential, reflectingchanges in mitochondrial membrane permeability, is another recognisedmethod of identification of apoptotic cells.

[0012] A number of other methods of identification of cells undergoingapoptosis and of apoptotic cells, many using monoclonal antibodiesagainst specific markers for apoptotic cells, have also been describedin the scientific literature.

[0013] Methods of quantifying apoptotic cells and apoptotic bodies in acellular composition are known and readily practiced by persons of skillin the art. Techniques include staining of the treated cell population,with an appropriate, selective dye such as fluorescein-conjugatedannexin V, followed by incubation and analysis by flow cytometry.

[0014] Necrosis, in contrast, is cell death of a pathological nature,resulting from injury, bacterial toxin effects, inflammatory mediators,etc., and involving membrane rupture and release of intracellularcontents to the surrounding tissue, often with harmful inflammatoryconsequences. Necrotic cells may be detected and characterized bydetection of compromised cell membranes e.g. by methods such as stainingwith propidium iodide followed by flow cytometry or microscopy.

SUMMARY OF THE INVENTION

[0015] According to the present invention, the administration ofapoptotic cells and/or apoptotic bodies previously prepared ex vivo, isused in the prophylaxis and/or treatment of medical disorders in whichthere is dysfunction of the cells of the endothelium, the cellularlining of blood vessels.

[0016] In one of its method aspects, this invention is directed to amethod for the treatment of or prophylaxis against an endotheliumdysfunction disorder in a mammalian patient, which comprisesadministering to the patient an effective amount of apoptotic bodiesand/or apoptotic cells.

[0017] These methods are preferably accomplished by administering to thepatient the pharmaceutical compositions described herein.

BRIEF DESCRIPTION OF THE DRAWING

[0018] The FIGURE is a graph showing a comparison of net ear swelling inmice treated with the compositions of this invention and a controlgroup.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] This invention is directed to the treatment and/or prophylaxis ofendothelium dysfunction disorders by administering apoptotic cellsand/or bodies to a mammalian patient.

[0020] The endothelium is a cellular layer lining the walls of bloodvessels of a mammal. It is a highly specialized interface between bloodand underlying tissues and has a number of functions, including: controlof haemostasis by inhibiting platelet aggregation (antithrombotic andregulating the coagulation and fibrolinolytic systems); control ofvascular tone, and hence blood flow; control of blood vessel smoothmuscle growth; and selective permeability to cells and proteins.

[0021] Normally, the endothelium maintains vascular homeostasis byresponding to physiological stimuli, for example, changes in blood flow,oxygen tension etc., by adaptive alteration of function. Dysfunctionalendothelium has an impaired response to such physiological stimuli, andcan ultimately lead to medical disorders. A number of subsets ofendothelial dysfunction have been recognized, including EndothelialActivation, and Endothelial-mediated Vasodilatory Dysfunction [see DeCaterina (2000). “Endothelial dysfunctions: common denominators invascular disease”. Current Opinions in Lipidology 11:9-23].

[0022] Endothelial activation may lead to the initiation ofatherosclerosis and is a process whereby there is an inappropriateup-regulation and expression of cell attraction and cell adhesionmolecules on endothelial cells. This particularly involves theMacrophage Chemoattractant Protein-1 (MCP-1), chemoattractants forlymphocytes (IP-10, MIG, I-TAG), and the Vascular Cell AdhesionMolecule-1 (VCAM-1), to which the monocytes and lymphocytes adhere. Onceadherent, the leucocytes enter the artery wall. The monocytes andlymphocytes are recruited to the intima (sub-endothelial layers) of theblood vessels by these cell attraction and cell adhesion molecules ofthe activated endothelium during the early stages of atherosclerosis(see Libby, P. (2000) “Changing concepts of atherogenesis,” Journal ofInternal Medicine 247:349-358.)

[0023] Endothelial-mediated Vasodilatory Dysfunction is characterized bya reduction or loss of endothelium-dependent vasodilation and involves“decreased nitric oxide bioavailability” (decreased production,increased destruction and/or decreased sensitivity to nitric oxide). [DeCaterina (2000), cited above]. Nitric oxide induces vasodilation byrelaxing the smooth muscle cells of the blood vessel wall.Endothelial-mediated Vasodilatory Dysfunction can be measured as areduction in vasodilation in response to acetylcholine, or as a reducedvasodilatory response following occlusion of arterial blood flow(reactive hyperaemia) for example using a sphygmomanometer cuff. As wellas leading to a reduction in vasodilation, decreased endothelial nitricoxide bioavailability can also result in an increase in the productionof vaso-constriction and hypertension. Platelet aggregation is inhibitedby nitric oxide, hence a decrease in nitric oxide bioavailability canlead to an increase in platelet aggregation and consequent thrombosis.These are just a few examples of how decreased nitric oxidebioavailability resulting from Endothelial-mediated VasodilatoryDysfunction can have pathological consequences.

[0024] The medical disorder resulting from endothelial dysfunction, andhence treatable in accordance with the present invention, can be acardiovascular disorder such as atherosclerosis, peripheral vasculardisease, congestive heart failure, stroke, myocardial infarction,angina, hypertension and the like. It can be a vasospastic disorder suchas Raynaud's disease, cardiac syndrome X, migraine and the like. It canbe the damage resulting from ischemia (ischemic injury orischemia-reperfusion injury). In summary, it can be substantially anydisorder that results from an inappropriately functioning endothelium.

[0025] “Apoptotic cells” and “apoptotic bodies,” as the terms are usedherein, means cells and cell bodies which exhibit one or more of thefollowing apoptosis-characterizing features: surface exposure ofphosphatidylserine, as detected by standard, accepted methods ofdetection such as Annexin V staining; alterations in mitochondrialmembrane permeability measured by standard, accepted methods (e.g.Salvioli, S., Ardizzoni, A., Franceschi, C. Cossarizza, A. (1997) “JC-1,but not DiOC6(3) or Rhodamine 123, is a Reliable Fluorescent Probe toassess Delta Psi Changes in Intact Cells: Implications for Studies onMitochondrial Functionality during Apoptosis,” FEBS Letters 411: 77-82];evidence of DNA fragmentation such as the appearance of DNA laddering onagarose gel electrophoresis following extraction of DNA from the cells[Teiger, E., Dam, T. V., Richard, L., Wisnewsky, C., Tea, B. S.,Gaboury, L., Tremblay, J., Schwartz, K. and Hamet, P. (1996) “Apoptosisin Pressure Overload-induced Heart Hypertrophy in the Rat,” Journal ofClinical Investigation 97; 2891-2897], or by in situ labeling (seeGavrieli et al., 1992, referenced above).

[0026] The compositions of apoptotic cells and/or apoptotic bodies foruse in the present invention preferably comprise not more than about 35weight percent of necrotic cells and/or necrotic bodies based on thetotal weight of the apoptotic cells/bodies and necrotic cells/bodies;more preferably, not more than about 20 weight percent; and even morepreferably, not more than about 10 weight percent. At these levels, thepresence of such necrotic cells and/or bodies are believed not tosignificantly alter in vivo processes. In its most preferred embodiment,the apoptotic cells/bodies are substantially free of necrotic cellsand/or bodies (i.e., less than about 2 weight percent of necroticcells/bodies).

[0027] The apoptotic cells and/or apoptotic bodies for use in thepresent invention are prepared ex vivo from mammalian cells that arecompatible with those of the mammalian patient. They can be preparedfrom substantially any type of mammalian cell including cultured celllines. Preferably they are prepared from a cell type derived from themammalian patient's own body or from an established cell line. Morepreferably they are prepared from white blood cells of blood compatiblewith that of the mammalian patient, more preferably from the patient'sown white blood cells and even more preferably from the patient's own Tlymphocytes. Even more preferably they are prepared from an establishedcell line. The apoptotic cells and/or apoptotic bodies are preparedextracorporeally prior to administration to the patient. Thus, in oneembodiment, an aliquot of the patient's blood may be withdrawn, e.g. byvenipuncture, and at least a portion of the white cells thereofsubjected extracorporeally to apoptosis inducing conditions.

[0028] A variety of methods of inducing apoptosis in mammalian cells, soas to create apoptotic cells and/or apoptotic bodies, are known in theart and essentially any of these can be adopted in preparing apoptoticbodies for use in the present invention. One such method is thesubjection of the cells to ionizing radiation (γ-rays, x-rays, etc.)and/or non ionizing electromagnetic radiation including ultravioletlight. Apoptosis can be induced by subjecting cells to ultrasound.

[0029] Another method is the treatment of the cells with drugs such asnon-specific protein kinase inhibitors as exemplified by staurosporine(see Bombeli, Karsan, Tait and Hirlan, (1997) “Apoptotic VascularEndothelial Cells Become Procoagulant”, Blood, Vol. 89:2429-2442). Also,certain chemotherapeutic agents used for the treatment of malignanttumours induce apoptosis, for example, adriamycin, as can statin drugs(3-hydroxy-3methylglutaryl coenzyme A reductase inhibitors) [Guijarro C,Blanco-Colio L M, Ortego M, Alonso C, Ortiz A, Plaza J J, Diaz C,Hernandez G, Edigo J (1998), “3-hydroxy-3methylglutaryl coenzyme Areductase and isoprenylation inhibitors induce apoptosis of vascularsmooth muscle in culture, Circulation Research 83: 490-500] andcolcicine [Suzuki Y (1998)”, “Cell death, phagocytosis and neurogenesisin mouse olfactory epithelium and vomeronasal organ after colcicinetreatment,” Annals of the New York Academy of Sciences 855: 252-254].The use of ligands for death receptors on cells, such as Fas-ligand,will be apparent for inducing apoptosis from the discussion of apoptosisabove. A further method is the application of oxidative stress to cellsextracorporeally (see for example Buttke and Sandstrom (1994) “OxidativeStress as a Mediator of Apoptosis,” Immunology Today, Vol. 15:7-10).This can be achieved by treating the cells, in suspension, with chemicaloxidizing agents such as hydrogen peroxide, other peroxides andhydroperoxides, ozone, permanganates, periodates, and the like.Biologically acceptable oxidizing agents are preferably used, so as toreduce potential problems associated with residues and contaminations ofthe apoptotic cells and/or apoptotic bodies so formed.

[0030] The present invention is not restricted to any particular methodof producing apoptotic cells and/or apoptotic bodies, for use herein,and any suitable, known process can be used.

[0031] Methods for the detection and quantitation of apoptosis can beused to determine the presence and level of apoptosis in the preparationto be administered to the patient in the present invention. A method asdescribed in the introduction above should be used to confirm the levelof apoptosis achieved prior to administration. They are suitablypurified prior to use, by methods known in the art, such as differentialcentrifugation.

[0032] In preparing the apoptotic cells and/or apoptotic bodies, careshould be taken not to apply excessive levels of oxidative stress,radiation, drug treatment, etc., since otherwise there is a significantrisk of causing necrosis of at least some of the cells under treatment.Necrosis causes cell membrane rupture and the release of cellularcontents often with biologically harmful results, particularlyinflammatory events, so that the presence of necrotic cells and theircomponents along with the apoptotic bodies is best avoided. Appropriatelevels of treatment of the cells to create apoptotic bodies for use inthe present invention depend to some extent on the nature of the chosencells and cellular composition, and the type of treatment chosen toinduce apoptosis. Such appropriate levels are readily determinable bythose skilled in the art, having regard to the available scientificliterature on the subject including the above-reference articles.

[0033] One preferred process according to the present invention involvesthe culture of cells from the patient, or a compatible mammalian cellline. The cultured cells may then be treated to induce apoptosis and tocreate apoptotic cells and/or apoptotic bodies therein. The cells,suspended in the patient's plasma or another suitable suspension medium,such as saline or a balanced mammalian cell culture medium, can then beadministered as indicated below. The numbers of apoptotic cells and/orbodies can be determined by published methods available in thescientific literature on the subject including the above-referencearticles. The numbers of such apoptotic cells and/or apoptotic bodiesrequired for administration to the patient to obtain the requiredclinical benefit will vary depending on the source of cells, thepatient's condition, the age and weight of the patient and otherrelevant factors which are readily determinable by the attendingclinician.

[0034] Another example of a preferred process according to the presentinvention accordingly involves extraction of an aliquot of blood fromthe patient to be treated, separation of the white cells therefrom,suspension of the white cells in plasma or another suitable suspensionmedium, such as saline or a balanced mammalian cell culture medium andtreatment of the white cells under apoptosis-causing conditions, e.g.with a chemical such as sodium butyrate, so as to create a cellularcomposition in which significant numbers of the white cells therein havebeen apoptosed so as to create therein substantial numbers of apoptoticcells or bodies. Then the treated composition is re-administered to thepatient. More preferably, T lymphocytes, isolated from the blood byknown means, and suspended as above, may be used as a source ofapoptotic cells and apoptotic bodies.

[0035] The number of viable cells selected for treatment to createapoptotic cells and/or apoptotic bodies is suitably up to about 4×10⁹,preferably from about 1,000,000 to about 1,000,000,000 and mostpreferably from about 50,000,000 to about 150,000,000, for eachadministration to a human patient. From about 10% to 90%, preferablyfrom about 30% to 70% of the cellular compositon for administration iscomprised of apoptotic cells and apoptotic bodies, the balance beingviable cell and necrotic cells. Accordingly, the preferred amounts ofapoptotic cells and/or apoptotic bodies for administration are thoseproduced by subjecting these numbers of cells to the apoptosingconditions. When whole blood is used as the source of the cells to besubjected to the apoptosis inducing conditions, these numbers of whitecells are obtainable in blood aliquots of volume up to about 400 mls,preferably up to 100 mls. More specifically, 50,000,000 to 150,000,000cells is equivalent to the white cells in blood aliquots of volume 10-30mls.

[0036] The volume of the aliquot of blood withdrawn from the patient fortreatment to create apoptotic cells and/or apoptotic bodies therein issuitable up to about 400 ml, preferably from about 0.1 to about 100 ml,and most preferably from about 5 to about 15 ml. Accordingly, thepreferred amounts of apoptotic cells and/or apoptotic bodies foradministration are those corresponding to the numbers derivable from thewhite blood cells, or isolated T lymphocytes, contained in suchquantities of whole blood, following subjection to apoptosis-inducingconditions.

[0037] The suspension of treated apoptotic cells and/or bodies foradministration to the patient is prepared in a biologically acceptableliquid suspending medium, such as the patient's serum or plasma, salineor balanced mammalian cell culture medium. The addition of otherfactors, such as cytokines, hormones, products of stressed cells orother appropriate biologically active material may enhance the benefitof the administered apoptotic cellular materials. The aliquot can bere-introduced into the patient's body by any suitable method, mostpreferably intramuscular injection but also including subcutaneousinjection, mini-grafting, intra-peritoneal injection, intra-arterialinjection, intravenous injection and oral administration. The apoptoticentities can be delivered to the specific body organ and/or site byusing any appropriate, known delivery system.

[0038] The compositions of this invention may optionally include apharmaceutically acceptable excipient. Some examples of suitableexcipients include sterile water, sterile saline, phosphate bufferedsaline, and the like.

[0039] When administered, the pharmaceutical compositions comprise aneffective amount of apoptotic bodies/cells to induce a suitableprophylactic and/or therapeutic response in the patient at risk ofsuffering or suffering from an endothelial dysfunction related disease.Preferably, the composition administered to the mammalian patientcomprises from about 10,000 to 10,000,000 apoptotic cells or bodies perkilogram of body weight, more preferably from about 500,000 to 5,000,000and most preferably from about 1,500,000 to 4,000,000 apoptotic cells orbodies per kg body weight. The specific dose employed will, of course,be dependent upon the age, weight and severity of the disease in thetreated patient all of which are within the skill of the attendingclinician.

[0040] For most effective treatment and prophylaxis of mammaliandisorders involving an endothelial dysfunction, the patient may be givena course of treatments with apoptotic cells and/or bodies according tothe invention. Each course of treatment may involve administration tothe patient of from 1 to 6 aliquots of suspended cellular material, asdescribed above. No more than one such aliquot should be administeredper day, and the maximum rest period between any two consecutiveadministrations should be not greater than about 21 days. Boostertreatments as described below may advantageously be used. To maintainthe desired effects, the patient may undergo booster treatments, with afurther course of administration of aliquots of suspended apoptoticcells and/or apoptotic bodies as described above, at intervals of threeto four months.

[0041] As noted, the present invention is applicable to the treatmentand/or prophylaxis of a wide variety of mammalian disorders that involveendothelial dysfunction. These include, but are not limited to,cardiovascular disease, such as atherosclerosis, peripheral vasculardisease, congestive heart failure, stroke, myocardial infarction,angina, hypertension, etc., vasospastic disorders such as Raynaud'sdisease, cardiac syndrome X, migraine, etc; and the damage resultingfrom ischemia (ischemic injury or ischemia-reperfusion injury). Insummary it can be substantially any disorder that results from aninappropriately functioning endothelium.

[0042] The invention is further described, for illustrative purposes, inthe following specific examples.

EXAMPLE 1

[0043] Experiments to demonstrate the invention were conducted onlaboratory mice, under approved conditions for conducting suchexperiments.

[0044] The effectiveness of the treatment according to a preferredembodiment of the present invention, on contact hypersensitivity (CHS),an example of a Th-1-cell inflammatory disorder which is known to bemediated by inflammatory cytokines, was assessed on laboratory mice,according to approved animal experimentation procedures, using themethod described by Kondo et. al., “Lymphocyte function associatedantigen-1 (LFA-1) is required for maximum elicitation of allergiccontact dematitis” Br. J. Dermatol. 131:354-359 (1994), with minorvariations. The disclosure thereof is incorporated herein by reference.Briefly, to induce CHS, the abdominal skin of each mouse was shaved andpainted with dinitrodifluorobenzene DNFB, the sensitizing chemical,using 25 μl of 0.5% DNFB in 4:1 acetone:olive oil solution. Thissensitization was applied to two groups of Balb/c mice, 10 animals intotal.

[0045] Apoptotic bodies were prepared from murine fibroblasts. Themurine fibroblasts were treated with 50 mM sodium butyrate in RPMImedium, at confluency for one day, and then the sodium butyrate mediumwas changed. To increase the number of apoptotic cells and bodies, thecells can additionally be irradiated with UV-light (e.g. 75 mj).Supernatant containing floating cells is removed 24 hours followingirradiation.

[0046] Apoptotic bodies were quantitated by centrifuging the supernatant(1200 rpm, 5 minutes), aspirating the supernatant, washing the resultingcell pellet with PBS and centrifuging again, as above. The pelletcontaining the apoptotic bodies was re-suspended in PBS. The cells werestored in PBS at 4° C. for the duration of the experiment. The cells tobe stained for quantitation were re-suspended in 1× binding buffer at aconcentration of 1×10⁶ cells/ml. 100μl of the cells were transferred toa 5 ml tube, and 10 μl of fluorescein-conjugated annexin V and 10 μlpropidium iodide reagent was added. The cells were gently vortexed andthe cell mixture incubated for 15 minutes at 25° C. in the dark.Following the incubation, 400 μl of 1× binding buffer was added to eachtube. The sample was analyzed on a flow cytometer over one hour.

[0047] Of the two groups of sensitized mice, the first, control group A,received no treatment. The second, test group B, was treated with aninjection of suspended apoptotic bodies prepared as described above, 50μl volume containing at least 150,000 bodies per injection of bloodsubjected to stressors as described above. Treatments, each involvingintramuscular injection of 50 μl of the respective liquid, started onthe day of sensitization and were repeated every day for a total of sixdays. On the same day as the last treatment, but after itsadministration, the animals were challenged with DNFB, by applying tothe right ear of each animal 10 μl of 0.2% solution of DNFB in acetoneand olive oil. To the left ear of each animal was applied theacetone/olive oil solvent, without DNFB. Inflammation due to CHSmanifests itself in a swelling of the right ears. Ear thickness wasmeasured, 24 hours after challenge, with a Peacock spring-loadedmicrometer (Ozaki Co., Tokyo, Japan). The results were expressed as thethickness and difference in thickness of the right ears and the leftears of each animal, at 24 hours after challenge.

[0048] The experiments were repeated, using more sets of two groups ofanimals, a sufficient number of times to ensure statistical significancein the results. A notable and significant reduction in ear thickness(inflammation) was observed with the animals treated with the apoptoticcells and apoptotic bodies suspension in accordance with the invention,as compared with the untreated group, demonstrating a significantreduction in inflammation. The results are presented in the followingTable, and on the accompanying FIGURE, as a bar graph of net earswelling (difference between right ear and left ear thickness), for eachgroup, with “standard deviation” shown by the vertical line at the topof each column. TABLE 1 Group Left ear Right ear Difference A 17 31 14 A18 39 21 A 17 30 13 A 18 32 14 A 18 31 13 Mean: 15 S.D: 3.391165 B 21 3110 B 18 18  0 B 17 30 13 B 20 24  4 B 18 22  4 Mean: 6.2 S.D.: 5.215362

[0049] An analysis of the suspension of apoptotic cells and bodiesadministered to the animals of test group B indicated the presencetherein of approximately 40% apoptotic cells and bodies, balance viablecells and minor amounts of necrotic cells (not more than 20%), thepresence of which is believed not to be significant in the in vivoprocess.

EXAMPLE 2

[0050] The above test procedure was repeated on similar groups ofanimals, a control group and a test group, but using a suspension ofapoptotic cells and bodies on the test group which comprised about 60%apoptotic cells and bodies, balance viable cells and a minor amount (notmore than 20%) of necrotic cells. Essentially similar results wereobtained.

[0051] The effectiveness of the processes and compositions of thepresent invention in preventing and alleviating inflammation due to CHSindicates that administration of apoptotic cells and bodies as describedup-regulates the in vivo generation of anti-inflammatory Th-2 derivedcytokines such as IL-10 (known to be implicated in CHS—see Kondo,McKenzie and Sauder, “The Journal of Investigative Dermatology,” Vol.103, 1994, page 811-814) and/or down-regulates Th-1 inflammatorycytokines such as TNFγ, IL-6 and IL-12. These inflammatory cytokines areimplicated in endothelial dysfunctions which manifest themselves ascardiovascular disorders, such as atherosclerosis, peripheal vasculardisease, congestive heart failure, stroke, myocardial infarction,angina, hypertension and the like; vasospastic disorders such asRaynaud's disease, cardiac syndrome X, migraine and the like; and damageresulting from ischemia (ischemic injury or ischemia-reperfusioninjury). Consequently, the finding of success in CHS treatment reportedin the above Examples is indicative of successful use of the process andcompositions in the treatment and prophylaxis of a wide variety ofendothelial dysfunction disorders including those discussed above.

What is claimed is:
 1. The use of apoptotic bodies and/or apoptoticcells in treatment and/or prophylaxis in mammalian patients of medicaldisorders resulting from or involving endothelial dysfunction.
 2. Theuse of apoptotic bodies and/or apoptotic cells in the preparation of amedicament for the treatment and/or prophylaxis of medical disordersresulting from or involving endothelial dysfunction in mammalianpatients.
 3. The use of claim 2 wherein the apoptotic bodies and/orapoptotic cells are in a liquid suspension along with viable cells. 4.The use of claim 3 wherein the apoptotic bodies and/or apoptotic cellscomprise from 10% to 90% of the cellular portion of the suspension. 5.The use of claim 4 wherein the apoptotic bodies and/or apoptotic cellscomprise from 30% to 70% of the cellular portion of the suspension. 6.The use of claim 1 or 2 wherein the apoptotic bodies and/or cells arederived from extracorporeal treatment of blood cells compatible withthose of the mammalian patient.
 7. The use of claim 1 or 2 wherein theapoptotic bodies and/or cells are derived from established cultured celllines.
 8. Thu use of claim 6 wherein the blood cells are white bloodcells of blood compatible with that of the mammalian patient.
 9. The useof claim 8 wherein the blood cells are the patient's own white bloodcells.
 10. The use of claim 9 wherein the blood cells are the patient'sown T lymphocytes.
 11. The use of claim 1 or 2 wherein the disorder isselected from the group consisting of atherosclerosis, periphealvascular disease, congestive heart failure, stroke, myocardialinfarction, angina, hypertension, Raynaud's disease, cardiac syndrome X,migraine, ischemic damage, inflammatory bowel disease and graft versushost disease.
 12. The use of claim 1 or 2 further comprisingadministering to a human patient a dosage of apoptotic bodies and/orapoptotic cells comprising from 10,000 to 10,000,000 apoptotic bodiesand/or apoptotic cells per kilogram body weight of the patient.
 13. Theuse of claim 12 wherein the dosage contains from 500,000 to 5,000,000apoptotic bodies and/or apoptotic cells per kilogram body weight of thepatient.
 14. The use of claim 12 wherein the dosage contains from1,500,000 to 4,000,000 apoptotic bodies and/or apoptotic cells perkilogram body weight of the patient.
 15. A method for treatment of orprophylaxis against medical disorders resulting from or involvingendothelial dysfunction in a mammalian patient, which comprisesadministering to the patient an effective amount of apoptotic bodiesand/or apoptotic cells.